In general, spectroscopic systems allow for the determination of the spectral (i.e., wavelength) composition of an object or a scene. Generally, these systems collect the total light coming from or emitted by the object. The wavelengths that comprise the collected light are typically separated with the use of a dispersive element employing refractive means such as a prism or diffractive means (such as, for example, a grating). After being reflected by or transmitting through one of these dispersive elements, the different wavelength components of the light propagate in different directions, and their intensities are recorded by a one-dimensional array of detector pixels. While standard spectrometers are excellent devices for determining the spectral composition of all the light emanating from an object, they are typically unable to provide two dimensional spatial maps of the spectra emanating from an object.
The present invention generally falls within the category of imaging spectrometers. Imaging spectrometers are more sophisticated than standard spectrometers because they allow for the measurement of the wavelength composition of light coming from each point in the object. Imaging spectrometers providing high spectral resolution (i.e. less than 2 nm) are known in the art as either “spectral scanning” or “spatial scanning” systems.
Spectral scanning systems typically take a series of images, where each image represents a full field-of-view, two-dimensional representation of the object, comprised of light within a certain spectral bandpass. Separate wavelength images are taken one after the other, or sequentially in time. Specific systems for spectral scanning include those incorporating liquid crystal tunable filters (LCTF), acoustic optical tunable filters (AOTF), and interferometric systems such as the Fourier transform spectrometer (FTS) and the Fabry-Perot spectrometer (FPS).
With LCTF-based systems, the properties of the liquid crystals are adjusted to “tune” the spectral bandpass of the filter. In this way, different, full field-of-view, spectral images are obtained over time. These systems have a number of disadvantages. For example, these systems are polarization-sensitive and, as a result, they have low transmission efficiency, resulting in significant light loss. As another example, the minimum spectral bandpass is usually 10 nm or greater. This is the result of the physics of the LCTF and a practical limitation imposed by the significant light loss. Therefore, high-spectral resolution (less than 2 nm) imaging is rarely possible with these systems. When AOTFs are used as tunable bandpass filters, AOTF-based systems have similar disadvantages.
An FTS system is usually based on the design of a Twyman-Green interferometer or a Sagnac interferometer (such as disclosed in U.S. Pat. No. 5,539,517), both of which are typically used to ascertain the spectral content of a point source. In typical operation for this type of system, a positive lens collimates the light from the point source before it enters the interferometer. Either a test arm or reference arm mirror is scanned along the optical axis with the intensity being detected at each scan position. Taking the inverse Fourier transform of the envelope of the detected signal yields the spectral intensity of the object as a function of frequency or wavelength.
An FPS system is based on another interferometric design that generally employs two highly reflective mirrors to form an optical cavity that functions as a spectral filter. In this type of a system, collimated light entering this system will undergo multiple reflections within the optical cavity. As a result of this configuration, only the particular wavelength component for which all the multiple reflections interfere constructively will pass through the optical cavity to be recorded by a detector. The particular wavelength that is passed by the optical filter depends on the distance between the two highly reflective mirrors. As this distance is changed, the wavelength passed by the filter also changes. Thus, the spectral bandpass of the FPS system is a function of the lateral separation of the mirrors. In this way, as one mirror is scanned along the optical axis, effectively changing the distance between the mirrors, the spectral bandpass is changed and the different spectral components of the source are recorded sequentially by the detector.
The FTS and FPS systems are also capable of performing imaging spectrometry and determining the spectral composition of an object on a point-by-point basis. However, there are certain limitations imposed by the physical geometry of these systems. In addition, in both cases, the system field of view is restricted. For example, with regard to the FTS system, the length of the system, combined with the small size of the mirrors, restricts the field of view because optical rays will not propagate through the system for large angles. Therefore, the number of points on an object which can be acquired is limited. Another problem arises with respect to image registration. Two-dimensional images are acquired as one of the mirrors is scanned. Problems associated with scanning, such as mirror jitter, uneven scanning, or beam-walking, create registration problems between the images in the different spectral bands. With regard to the FPS system, it is also limited to a small field of view because of two main effects. For example, the light coming from the source undergoes multiple reflections within the mirrored optical cavity before emerging from the system. When the incident light comes from an off-axis point on the object, it enters the cavity at an incident angle other than zero. Consequently, as the light undergoes multiple reflections, it will walk along the mirrors and eventually leak out of the cavity. The result of this behavior is that as the field increases, the light throughput of the system decreases. Another problem with the FPS configuration has to do with spectral bandpass variation with field. Since the effective mirror separation changes with field angle, so does the spectral bandpass. To minimize this field-dependent spectral variation, the field of view must necessarily be small.
Typically spectral scanning systems suffer from the fact that their inherent design does not allow for confocal imaging of the object. Confocal imaging systems always require some sort of spatial scanning. Spectral scanning systems have no spatial scanning attribute, making confocal microscopy impossible.
In addition, all spectral scanning systems have a fundamental flaw when used in low light applications such as fluorescence microscopy. These applications suffer from a phenomenon called photobleaching, where the fluorescence of an object decreases with the length of the exposure, and phototoxicity, where the light that is used to illuminate the object is toxic to the object. In all spectral scanning systems the entire object must be illuminated the entire time the wavelength scan is taking place. As a result, the images acquired later in the sequence (i.e. the longer wavelength images) will be dimmer than the images acquired first because of photobleaching. This change in intensity is not something that can easily be corrected after acquisition of the images. If toxicity becomes an issue, then the object's characteristics will change over time as the object becomes damaged. This effect cannot be corrected for post acquisition.
Spatial scanning systems, achieve the same result as the spectral scanning systems, but without the drawbacks discussed. Spatial scanning systems are optical imaging systems where a portion of a two-dimensional object is imaged onto a detector. Spatial scanning systems are usually classified into two types, based on the dispersion mechanism used. There are prism-based systems (such as disclosed in U.S. Pat. No. 5,127,728) and grating-based systems. In typical spatial scanning systems, a dispersive element in the optical path spreads the wavelength components of each point in the image along one dimension of a detector. This behavior effectively creates a series of rainbows on the detector. When a prism is used, the dispersion is achieved via refraction of the light. Alternatively, when a grating is used, the dispersion is achieved via diffraction of the light. Prism-based systems have the advantage of higher light throughput than grating-based systems. However, prism-based systems have the significant disadvantage of producing non-linear dispersion, requiring data correction via interpolation for proper visualization and processing. Since the dispersion effect via refraction is not as pronounced as it is via diffraction, prism-based systems generally require longer optical trains to achieve the same unit dispersion as grating-based systems. Grating-based systems display linear dispersion, eliminating the data correction step. The dispersion is higher than prism-based systems, which makes the optical trains shorter, but the efficiencies slightly lower than prism-based systems.
Gratings can be reflective or transmissive. Reflective gratings that utilize metal coatings for their reflectivity are used often for imaging spectrometers in a design configuration known as the Czerny-Turner configuration. This configuration utilizes a symmetric optical design with two spherical mirrors and a planar reflective grating. The symmetry of the design minimizes optical aberration's; however, to obtain a reasonable field-of-view, the optical train tends to be long, preventing the development of a compact system. Furthermore, the reflective gratings used in the Czerny-Turner configurations are usually made with metal coatings that are quite polarization sensitive, meaning P-polarized light will yield a different result than S-polarized light. This causes a significant problem in fluorescence microscopy applications that are often, by their nature, highly polarization-dependent processes. Transmission gratings and reflective gratings that utilize “polarization insensitive” coatings do not present the same problems because they are not significantly polarization sensitive. As disclosed in the present invention, more compact designs can be utilized to obtain sizable fields of view.
In order to achieve spatial scanning, stages (such as motorized stages, for example) are used to move the object from one position to the next to create a two dimensional image. In some instances, the object is held fixed while the illumination is scanned and the detection aperture is scanned in conjunction with the detector (such as disclosed in U.S. Pat. No. 6,166,373). These approaches are generally very slow and do not have the necessary accuracy or temporal resolution for live-cell microscopy imaging applications.
Finally, all of these high-spectral resolution imaging systems are only capable of providing high-spectral resolution with low temporal resolution. In many instances, it is desirable to use high-spectral resolution to identify an optimal smaller set of wavelengths which are then acquired instantaneously on a single or multiple detectors (i.e. high temporal resolution). One example of the usefulness of a single, multi-mode spectral imaging system in microscopy is discussed in “Spectral imaging and its applications in live cell microscopy”, Timo Zimmerman, Jens Rietdorf, and Rainer Pepperkok, FEBS Letters 543, Advanced Light Microscopy Facility and Cell Biology/Cell Biophysics Programme, Heidelberg, Germany (May 2003) pages 87–92.
While different methods may be used to achieve imaging spectrometry, the prior art is generally not capable of providing the required performance within a compact, modular, flexible and fast system. The prior art also fails to disclose providing multiple imaging modalities within a single system.
There is a significant need for a compact, modular, flexible and fast system, which is adapted to provide different imaging modalities within a single system for microscopy. The present invention satisfies this need.